Effects of lotus root (the edible rhizome of Nelumbo nucifera) on the deveolopment of non-alcoholic fatty liver disease in obese diabetic db/db mice

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. The discovery of food components that would ameliorate NAFLD is therefore of interest. Lotus root, the edible rhizome of Nelumbo nucifera, contains a high level of polyphenolic compounds, and several health-promoting properties of lotus root have been reported. The present study examines whether dietary lotus root powder can protect db/db mice from hepatic injury. After 3 weeks of feeding, the hepatomegaly, hepatic triglyceride accumulation, and elevated hepatic injury markers in the serum were markedly alleviated in the Lotus diet-fed db/db mice relative to the control mice. These effects were partly attributable to suppression of the lipogenic enzyme activities and mRNA expression by the Lotus diet. The serum levels of adiponectin, which has been reported to have a protective effect against NAFLD, were significantly higher in the Lotus group than in the Control group of the db/db mice. Moreover, the hepatic expression of such inflammatory genes as tumor necrosis factor-alpha and monocyte chemoattractant protein-1 were markedly suppressed by the Lotus diet. We speculate that the development and progression of NAFLD were prevented by suppressing the expression of lipogenic and inflammatory genes as a result of the higher serum adioponectin level in the Lotus diet-fed db/db mice.     

Expression of Aeromonas caviae polyhydroxyalkanoate synthase gene in Burkholderia sp. USM (JCM15050) enables the biosynthesis of SCL-MCL PHA from palm oil products

AIMS: Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater is capable of utilizing palm oil products and glycerol to synthesize poly(3-hydroxybutyrate) [P(3HB)]. To confer the ability to produce polymer containing 3-hydroxyhexanoate (3HHx), plasmid (pBBREE32d13) harbouring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae (phaC(Ac)) was transformed into this strain. Methods and Results: The resulting transformant incorporated approximately 1 ± 0·3 mol% of 3HHx in the polymer when crude palm kernel oil (CPKO) or palm kernel acid oil was used as the sole carbon source. In addition, when the transformed strain was cultivated in the mixtures of CPKO and sodium valerate, PHA containing 69 mol% 3HB, 30 mol% 3-hydroxyvalerate and 1 mol% 3HHx monomers was produced. Batch feeding of carbon sources with 0·5% (v/v) CPKO at 0 h and 0·25% (w/v) sodium valerate at 36 h yielded 6 mol% of 3HHx monomer by controlled-feeding strategies. CONCLUSIONS: Burkholderia sp. USM (JCM15050) has the metabolic pathways to supply both the short-chain length (SCL) and medium-chain length (MCL) PHA monomers. By transforming the strain with the Aer. caviae PHA synthase with broader substrate specificity, SCL-MCL PHA was produced. Significance and Impact of the Study: This is the first study demonstrating the ability of transformant Burkholderia to produce P(3HB-co-3HHx) from a single carbon source.     

Partial purification and property of pyridoxine (pyridoxamine)-5′-phosphate oxidase isozymes from wheat seedlings

Isozymes of pyridoxine (pyridoxamine)-5′-phosphate oxidase (EC 1.4.3.5) were isolated from the extract of wheat seedlings by column chromatographies. From DEAE-Sephadex A-50, two fractions having pyridoxine-5′-phosphate oxidase activity were separated by eluting with ~0.075 and ~0.125 m phosphate buffers (pH 8.0). These fractions were further fractionated on a Blue-Sepharose CL-6B column, from which again two activities were eluted by 1.0 m KCl solution. One fraction, designated as E-I, used only pyridoxine 5′-phosphate as substrate, whereas the other, designated as E-II, oxidized not only pyridoxine 5′-phosphate but also pyridoxamine 5′-phosphate with approximately equal rates. The mobility on polyacrylamide disc gel electrophoresis and the substrate specificity of these two fractions were different. Therefore, they were concluded to be isozymes.     

The calcium-binding property of equine lysozyme

It was found that equine lysozyme binds one Ca2+. It was eluted with equimolar Ca2+ from a Bio-Gel P-4 column. Human lysozyme did not behave similarly. Equine lysozyme is concluded to be a calcium metallo-protein like alpha-lactalbumin, which is a homologue of hen egg white lysozyme.     

The first crystal structure of hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum

The extremely thermostable NAD-dependent glutamate dehydrogenase (NAD-GluDH) from Pyrobaculum islandicum, a member of the Crenarchaeota, was crystallized, and its 3D structure has been determined by X-ray diffraction methods. The homohexameric structure of Pb. islandicum glutamate dehydrogenase (Pis-GluDH) was solved and refined at a resolution of 2.9A with a crystallographic R-factor of 19.9% (Rfree 26.0%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. The secondary structural elements and catalytically important residues of the enzyme were highly conserved among the NAD(P)-dependent GluDHs from other sources. A structural comparison of Pis-GluDH with other NAD(P)-dependent GluDHs suggests that a significant difference in the alpha8-loop-alpha9 region of this enzyme is associated with its coenzyme specificity. From the analysis of the 3D structure, hydrophobic interactions between intersubunits were found to be important features for the enzyme oligomerization. It has been reported that Pis-GluDH is highly thermostable, like the GluDH of the hyperthermophilic archaeum Pyrococcus furiosus, and the increase in the intersubunit ion pair networks is responsible for the extreme thermostability of the Pc. furiosus enzyme. However, the number of intersubunit ion pairs in the Pis-GluDH molecules is much smaller than those of the Pc. furiosus GluDH. The number of hydrophobic interactions at the intersubunit interfaces were increased and responsible for the extremely high thermostability. This indicates that the major molecular strategy for high thermostability of the GluDHs may be different for each hyperthermophile.     

Experimental studies on the effects of tension on intraneural microcirculation in sutured peripheral nerves.

Tension on an end-to-end nerve suture causes injury to the intraneural vessels. Impairment of the transverse anastomoses occurs first, followed by degeneration of the intrafascicular vessels. The the transverse anastomoses undergo complete degeneration, and the extrafascicular vessels also sustain severe damage. This series of circulatory disturbances is an important factor to consider when determining the advisability of an end-to-end nerve suture.     

Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor

The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 Å resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs.Aeromonas species are Gram-negative facultative anaerobic bacteria found ubiquitously in a variety of aquatic environments (1). The main syndrome caused by infection with Aeromonas is gastroenteritis (2, 3), although, in severe cases, sepsis is induced as a deuteropathy (4, 5). Two species, Aeromonas hydrophila and Aeromonas sobria, are associated with human disease (6, 7). Factors thought to play important roles in the pathogenesis include fimbrial and afimbrial adherence factors; a variety of exotoxins, including hemolysin, cytotonic enterotoxin, heat-stable enterotoxin, and heat-labile enterotoxin; and several secreted proteases and lipases (8–12). Recently, we purified a 65-kDa A. sobria serine protease (ASP)² from the culture supernatant of A. sobria and found that the enzyme induces vascular leakage, reduces blood pressure through activation of the kallikrein/kinin system (13), promotes human plasma coagulation through activation of prothrombin (14), and causes the formation of pus and edema through the action of anaphylatoxin C5a (15). From these observations, we concluded that ASP mediates the induction of disseminated intravascular coagulation through α-thrombin production, which is a common and deadly consequence of sepsis (14).ASP is a kexin-like serine protease belonging to the subtilisin family (subtilases) (16), which can be subdivided into six groups: the subtilisins, thermitases, proteinase K, lantibiotic peptidases, pyrolysins, and kexins. Among the kexins, the first identified was Kex2 (17), which is expressed by Saccharomyces cerevisiae; subsequently, the mammalian kexin-like protease furin was identified (18). Furin processes the precursors of biologically active peptides and proteins via limited proteolysis at paired basic amino acids to generate biologically active molecules (19). The domain structures of kexins and furins include a signal peptide, a partially conserved propeptide, a highly conserved subtilisin-like domain containing the characteristic Asp, His, and Ser catalytic residues, a relatively well conserved region called the P-domain, and a transmembrane domain followed by a cytoplasmic tail (20–22). Kex2 usually shows a high degree of specificity for cleavage after dibasic (P2-P1: Lys-Arg or Arg-Arg) or multiple basic residues (23). Among the kexins, which are nearly all eukaryotic and share a substantial degree of sequence homology (>40%), ASP is positioned as the most distant member of this family on the phylogenetic tree (16). The domain structure of ASP consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. For maturation of ASP, the first 24 residues of the propeptide are cleaved, and although a functional P-domain is reportedly necessary for maturation of the subtilisin domain in kexins (24, 25), the function of the P-domain in ASP remains unknown. Notably, in an earlier study of ASP expression, we found that for the maturation of the ASP subtilisin domain, another gene product, encoded by open reading frame 2, is required to serve as a chaperone in the periplasmic space (26).Here, we report the crystal structure of wild-type ASP as a sepsis-related factor at 1.65 Å resolution. We found that ASP has a unique occluding region at the active site within the subtilisin domain and that a different form of ASP that is cleaved within the occluding region shows a different pattern of proteolysis from the native enzyme. Our findings suggested that the novel occluding region plays an important role in determining substrate specificity and that because it is unique, it could facilitate development of novel antisepsis drugs that have no inhibitory effect on furin-like human proteases.     

Specific In Situ Visualization of Plasma Cells Producing Antibodies against Porphyromonas gingivalis in Gingival Radicular Cyst: Application of the Enzyme-Labeled Antigen Method

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.